To ensure the accurate segregation of chromosomes during meiosis, recombination is used to create a crossover between homologous chromosomes. Recombination is initiated by the formation of programmed DNA double-strand breaks that are repaired through an interaction with a homologous chromosome, culminating in the formation of a joint DNA molecule. Models of recombination and proposed structures of the joint molecule are primarily based on the detection of recombination intermediates in yeasts, in vitro biochemical assays, and genetic data that is difficult to interpret because of overlapping gene functions. Models based on these data are often presented as universal facts for all organisms, when in fact there is little in vivo data available outside of yeasts. In this proposal,I describe experiments that will determine the relative contributions of different recombination models to meiotic crossover and noncrossover formation. I will use the metazoan Drosophila melanogaster and a unique genetic assay that allows the recovery of spontaneous recombination events within a heteroallelic locus that can then be analyzed to determine the arrangement of heteroduplex DNA. I also propose whole genome analysis of recombination events using next generation sequencing. Drosophila has the advantage that it is the only known organism in which a putative double Holliday junction resolvase, MEI-9, that is required for 95% of all crossovers has been identified. By combining the proposed genetic experiments with the already known biochemical properties of MEI-9, I will be able to make strong conclusions about how recombination works in Drosophila. These experiments have the advantage of being carried out in the context of a metazoan germline, and of measuring spontaneous recombination events at higher resolution than other metazoan systems are able to. Results from these experiments will expand knowledge of recombination mechanisms to model systems other than fungi and are directly applicable to both human fertility issues and cancer.